Genetic Basis for Human Congenital Heart Disease[unreadable] [unreadable] A new initiative using a gene discovery approach with mouse chemical mutagenesis was launched in 2002 to recover novel mutations causing congenital heart defects. To screen for mutants with congenital heart defects, we developed the use of mouse fetal echocardiography as a high throughput noninvasive method for cardiovascular phenotyping fetal mice in utero. Over the course of 3 years, we scanned nearly 14,000 fetuse from nearly 500 families (defined by the G1 male), with an estimate of 10,000 genes scanned (500 X 20 genes/family), or approximately 0.5 genome equivalent. Through the course of the screen, we have recovered mutants with phenotypes that include all of the major congenital heart defects seen clinically, such as persistent truncus arteriosus, transposition of the great arteries, pulmonary atresia/stenosis, atrial and ventricular septal defects, single ventricle, heterotaxy syndrome, situs inversus, and other anomalies. The mutations in 15 families have been mapped, with 9 mutations identified. Surprisingly, 7 of the 14 mutants with structural heart defects show heart and limb anomalies, with 4 exhibiting defects in left-right patterning. 5 of the 9 mutations identified were entirely novel genes not previously known to be associated with structural heart defects. The body of mutations we have recovered suggest a key pathway in congenital heart defects involves the cilia and sonic hedgehog (Shh) signaling. Recent studies from several laboratories show the primary cilia plays a key role in transducing Shh signaling. Thus, the focal point for much of the present research in my laboratory revolves around understanding the role of the cilia in orchestrating events in cardiovascular development, and how it transduces and regulate sonic hedgehog signaling and other cell signaling pathways. The first of these studies to be completed is now in press in the Journal of Clinical Investigation, where our studies focused on a dynein mutation in dnahc5, a gene also often mutated in patients with primary ciliary dyskinesia (PCD). [unreadable] [unreadable] High Frequency Ultrasound Cardiovascular Phenotyping [unreadable] [unreadable] My laboratory has a strong research interest in developing the use of ultra high frequency ultrasound imaging for mouse fetal cardiovascular assessments. In our ENU mouse mutagenesis screen, we utilized a clinical ultrasound system, the Acuson Sequoia for in utero fetal mouse imaging with a 15 MHz transducer. From the data obtained from scanning nearly 13,000 fetuses, a large database of normative values was established for cardiac structure and hemodynamic function in embryos from E14.5 to E19.5 (term) (Yu et al., 2007). To examine the feasibility of quantitative cardiac assessments in younger embryos (E11.5 to E13.5), we have begun investigating the use of a commercially available (Visualsonics) ultra high frequency ultrasound imaging system. Using a 40 MHz transducer with the Visualsonics Vevo 660 ultrasound system, we obtained quantitative hemodynamic measurements from the Tie2Cre deleted floxed-KLF2 knockout mouse embryos and their control littermate embryos. These studies were carried out in collaboration with Dr. Mark Kahn (University of Pennsylvania), and they showed KLF2 expression in endothelial cells play an essential role in modulating vascular tone. Surprisingly, our ultrasound analysis indicated the modulation of vascular tone begins at E11.5 (prior to septation of the outflow tract and ventricles), and is essential for survival of the embryo (Lee et al., 2006). We have also used the Visualsonics Biomicroscope to characterize and quantitatively assess adult cardiac function, utilizing the recently developed ECG gated imaging algorithm known as EKV. EKV simulates a frame rate of 1000 frames/second, a temporal resolution that is unmatched by any clinical ultrasound system. This high frame rate allows accurate 2D visualization of the adult heart through its entire cardiac cycle the adult mouse heart usually beats at a rate of 450-550 beats per minute. Using EKV, we did a detailed study of a new mutant mouse model recovered in our ENU screen with hypertrophic cardiomyopathy (Leatherbury et al., 2007). [unreadable] [unreadable] Atlas of Mouse and Human Cardiovascular Development [unreadable] [unreadable] To facilitate analysis of complex structural heart defects in our mutant mouse models, we developed instrumentation and methodology for episcopic fluorescence image capture (EFIC). EFIC is a histological method where tissue autofluorescence is used to image the block face as paraffin embedded tissue is sectioned. This generates 2D image stacks in perfect registration (Rosenthal et al., 2003), thereby allowing digital resectioning of the specimen in any plane. Moreover, high resolution 3D reconstructions can be generated with ease. EFIC imaging has been invaluable in allowing complete diagnosis of structural heart defects in each mutant animal, even those with very complex structural heart defects. Using EFIC, we have created a Mouse Cardiovascular Development Atlas containing 2D images and 3D volumes of the embryonic and fetal mouse heart from E9.5 to term. Using EFIC imaging, we are also constructing an Atlas of the Human Embryo from Carnegie Stage 13 to stage 23. These studies are being carried out in collaboration with Dr. Kohei Shiota, using human embryos largely from the Kyoto Collection of Human Embryos. The goal of this project is to provide details of development of all of the major organ systems, with three embryos from each Carnegie stage being imaged using MRI followed by EFIC reconstruction. These studies will complement our mouse mutagenesis screen, allowing further mouse/human comparisons that may yield novel insights into the etiology of human congenital heart disease.[unreadable] [unreadable] Connexin43 and the Modulation of Cardiovascular Development [unreadable] [unreadable] My laboratory has a long standing interest in understanding the role of connexins in cardiovascular development. Our focus is on the gap junction gene, connexin43, as connexin43 knockout mice are known to die at birth due to outflow tract obstruction associated with conotruncal heart malformations and coronary vascular anomalies. Our studies have indicated these defects arise from a requirement for connexin43 in two extracardiac cell populations - the cardiac neural crest cells and the epicardial/proepicardial cells. Both cell lineages are extracardiac and must migrate into the heart to support normal heart and coronary vascular morphogenesis. Using a combination of epicardial explants and in vivo studies, we have found a profound disturbance of epicardial EMT associated with the coronary vascular defects in the connexin43 knockout mice. Our recent studies showed this entail an unexpected role for connexin43 in modulating the dynamic organization of the actin and tubulin cytoskeleton (Xu et al., 2006). Studies are under way using various connexin43-GFP fusion constructs and connexin43 deficient mouse embryonic fibroblasts together with TIRF microscopy, to identify the protein domains important in modulating crosstalk between connexin43 and the actin/tubulin cytoskeleton. Of importance to note is the fact that the C-terminus of the connexin43 protein has been shown to be have a tubulin binding domain, and has been postulated to function as tubulin capping protein. In parallel to these imaging studies, in collaboration with Dr. Chloe Bulinski, we are also pursuing biochemical analysis of connexin43-tubulin protein interactions to elucidate the role of connexin43 in modulating the tubulin cytoskeleton. Overall, these studies suggest the requirement for connexin43 in cardiovascular development may reflect an essential integration of connexin43 with the dynamic regulation of the cytoskeleton.